Bile salt hydrolase probe and method of making and using the same

Abstract

Probe embodiments for targeting, identifying, and isolating enzymes exhibiting BSH activity as well as devices and kits that use the probes are described herein. Methods of making and using the probes, devices, and kits are also described. In some embodiments, probes, devices, and kits for targeting, identifying, and isolating enzymes in a biological sample are disclosed. In some embodiments, compositions and methods of treatment using the probes, devices, and kits disclosed herein are described.

We claim: 
     
       1. A probe having a structure satisfying Formula I 
       
         
           
           
               
               
           
         
       
       wherein each of R 1 , R 2 , R 3 , and R 4  independently is hydrogen or —OR, wherein each R independently is hydrogen, a counterion that balances a negative charge on the oxygen atom of the —OR group, or heteroaliphatic; R 5  is hydrogen or aliphatic; the linker is aliphatic or heteroaliphatic; DM is (i) 
       
         
           
           
               
               
           
         
       
       or (ii) biotin; pDM is an alkyne or an azide; PM comprises a benzophenone group or a diazirine group; AM is an alkyne, an azide, an activated ester, a carboxylic acid, a halide, or an alkyl halide; n is an integer ranging from 0 to 10; and m is 0 or 1. 
     
     
       2. The probe of  claim 1 , wherein m is 1 and the linker is —(CH 2 ) p —; 
       
         
           
           
               
               
           
         
       
       wherein each p independently is as an integer ranging from 1 to 10, and each q independently is an integer ranging from 0 to 10. 
     
     
       3. The probe of  claim 1 , wherein the DM group is present and is 
       
         
           
           
               
               
           
         
       
       or biotin. 
     
     
       4. The probe of  claim 1 , wherein each of R 1 , R 2 , R 3 , and R 4  independently is hydrogen, —OH, or —O-M + , wherein M +  is Li + , K + , Na + , or NH 4   + . 
     
     
       5. The probe of  claim 1 , wherein the activated ester is an NHS-ester. 
     
     
       6. The probe of  claim 1 , wherein m is 0 and the DM group is present and is 
       
         
           
           
               
               
           
         
       
     
     
       7. The probe of  claim 1 , wherein m is 1 and both the PM group and the pDM group are present. 
     
     
       8. The probe of  claim 1 , wherein m is 1 and both the AM group and the pDM group are present. 
     
     
       9. The probe of  claim 1 , wherein the probe has a structure satisfying Formula IIA or IIB 
       
         
           
           
               
               
           
         
       
     
     
       10. The probe of  claim 1 , wherein the probe has a structure satisfying one or more of Formulas IIIA-IIIF 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein each p independently is an integer ranging from 1 to 10 and each q independently is an integer ranging from 0 to 20. 
     
     
       11. A probe having one of the following sctructures 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein q is an integer ranging from 0 to 10. 
     
     
       12. A method, comprising:
 exposing a subject or a sample to a probe according to  claim 1  for a time sufficient to allow the probe to bind to an enzyme capable of hydrolyzing a bile salt to thereby form a probe-enzyme conjugate; and 
 analyzing the subject or the sample using a detection technique sufficient to identify a detectable signal produced by reaction between the probe and the enzyme. 
 
     
     
       13. The method of  claim 12 , wherein a detectable moiety is released from the probe upon binding of the enzyme to the probe. 
     
     
       14. The method of  claim 12 , wherein the probe comprises a pDM group and the method further comprises exposing the probe-enzyme conjugate to a detectable moiety group comprising an azide or an alkyne. 
     
     
       15. The method of  claim 12 , wherein the probe comprises a PM group and the method further comprises exposing the sample or the subject to a light source to facilitate binding of the PM group to the enzyme to thereby form the probe-enzyme conjugate. 
     
     
       16. The method of  claim 12 , further comprising performing genomic or proteomic assays using the probe-enzyme conjugate or microbes comprising the probe-enzyme conjugate. 
     
     
       17. A method, comprising:
 labeling at least one enzyme capable of hydrolyzing a bile salt with a probe according to  claim 1  to provide at least one labeled enzyme; 
 determining the presence of the at least one labeled enzyme in a sample by detecting a detectable signal; 
 sorting or isolating the at least one labeled enzyme or microbes comprising the at least one labeled enzyme; 
 identifying the microbes comprising the at least one labeled enzyme; 
 selecting a physical environment for altering the bile salt hydrolase activity of the at least one enzyme; and 
 altering bile salt hydrolase activity in the selected physical environment to thereby treat the disease or condition by enriching the selected physical environment with identified microbes, or reducing the amount of identified microbes in the selected physical environment, or reducing the bile salt hydrolase activity in the selected physical environment, or a combination thereof. 
 
     
     
       18. A kit, comprising:
 a substrate; and 
 a probe having a structure satisfying Formula I 
 
       
         
           
           
               
               
           
         
       
       wherein each of R 1 , R 2 , R 3 , and R 4  independently is hydrogen or —OR, wherein each R independently is hydrogen, a counterion that balances a negative charge on the oxygen atom of the —OR group, or heteroaliphatic; R 5  is hydrogen or aliphatic; the linker is aliphatic or heteroaliphatic; DM is a fluorophore, a chromogen, or biotin; pDM is an alkyne or an azide; AM is an alkyne, an azide, an activated ester, a carboxylic acid, a halide, or an alkyl halide; n is an integer ranging from 0 to 10; and m is 1; and wherein the probe is covalently bound to a surface of the substrate via the AM group. 
     
     
       19. A probe having a structure satisfying Formula I 
       
         
           
           
               
               
           
         
       
       wherein each of R 1 , R 2 , R 3 , and R 4  independently is hydrogen or —OR, wherein each R independently is hydrogen, a counterion that balances a negative charge on the oxygen atom of the —OR group, or heteroaliphatic; DM is 
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       or biotin; and n is an integer ranging from 0 to 10. 
     
     
       20. A probe having one of the following structures: